Imre I. Krizmanić

Population Systems of the Pelophylax esculentus Complex in the Southern Part of Its Range

Abstract. We explored the population systems of Pelophylax esculentus at the southern border of the distribution range for this complex. We used both morphological data and allozyme markers to describe population systems in this region. All three taxa in the complex, P. ridibundus, P. lessonae and their hybridogenetic form, P. kl. esculentus occurred in the research area. In four localities that were analysed near the Danube River, we found complex population systems of both the parent species and their hybridogenetic form. Southward from the border defined by the Sava and Danube riverbanks, only the P. ridibundus — P. kl. esculentus (R-E) system was detected. Among these R-E populations, the abundance of the hybridogenetic form declines sharply from the north to the south. The most common and widespread system in Central Europe, the P. lessonae — P. kl. esculentus population system, was detected in only one locality near the Sava River.

Comparative study of oxidative stress parameters and acetylcholinesterase activity in the liver of Pelophylax esculentus complex frogs

Comparative activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), the phase II biotransformation enzyme glutathione-S-transferase (GST), the concentrations of total glutathione (GSH), sulfhydryl groups (–SH) and the activity of the neurotoxicity biomarker acetylcholinesterase (AChE) were investigated in the livers of species belonging to the Pelophylax esculentus “complex” (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax kl. esculentus) from the wetland, Obedska bara in Serbia. The condition factor (CF) and hepato somatic index (HSI) were also calculated. All three species were caught at same locality and were exposed to the same environmental conditions. Liver SOD activity was lower in P. ridibundus than in P. kl. esculentus and P. lessonae; higher activities of CAT, GR and GST were observed in P. kl. esculentus frogs as compared to their parental species. The activity of GSH-Px was significantly lower in P. kl. esculentus. The activity of AChE was increased in P. lessonae as compared to P. kl. esculentus and P. ridibundus. Similar concentrations of GSH and —SH groups were observed in all investigated species. P. kl. esculentus had a higher CF, while the HSI was lower when compared to the parental species. Our findings suggest that the parental species (P. ridibundus and P. lessonae) possess more similar antioxidative responses to environmental conditions than the hybrid species P. kl. esculentus. The obtained results improve our understanding of the biology and physiology of these three closely related species.

Biomarkers of oxidative stress and metal accumulation in marsh frog (Pelophylax ridibundus)

To understand the effect of metals on the marsh frog Pelophylax ridibundus and the possible environment-induced changes in oxidative stress enzymes, we determined the concentrations of 18 metals: Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, Ga, Hg, In, Li, Mn, Ni, Pb, Sr, and Zn, in the tissues (liver, skin, and muscle) and water samples collected from different locations in Serbia. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST), acetylcholinesterase (AChE), and changes in concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH) were analyzed in the tissues of the sampled frogs. The concentrations of Al, Cd, Co, Cr, Cu, Fe, Ga, Hg, and Ni were highest in the liver, whereas those of Ba, Ca, Li, Mn, Pb, Sr, and Zn were highest in the skin. Hg correlated positively with liver SOD (in frogs from Danube-Tisza-Danube Canal (DTD)), muscle CAT (DTD), and muscle GST Ponjavica River (PO); Pb demonstrated a strong positive correlation with liver GR in frogs from Mt. Fruška Gora (FG); Cd only exhibited a positive correlation with AChE in the skin of frogs from DTD. In the skin, Zn correlated positively with AChE (DTD), SH groups (PO), and CAT (FG), and negatively with CAT, GST, and SH in the liver of frogs from DTD. Examination of these oxidative stress biomarkers, together with analysis of metal accumulation in the liver and skin of marsh frogs, provides a powerful tool for the assessment of metal pollution.

Antioxidative responses of the tissues of two wild populations of Pelophylax kl. esculentus frogs to heavy metal pollution

Heavy metal pollution of the aquatic environment is of great concern worldwide. Heavy metals are capable of inducing oxidative stress by increasing the formation of reactive oxygen species (ROS), and directly affecting the antioxidant defense system (AOS) in living organisms. The frog Pelophylax kl. esculentus is a semiaquatic species with semipermeable skin and a complex lifecycle, and represents a potentially useful bioindicator organism. The aim of this study was to investigate the accumulation of several heavy metals (Cd, Co, Cr, Cu, Fe, Hg, Ni, Pb and Zn), and their effects on selected parameters of the AOS, including the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), phase II biotransformation enzyme glutathione-S-transferase (GST), the total glutathione (GSH) contents and sulfhydryl (SH) group concentrations, as well as cholinesterases (ChEs) activities in the liver, skin and muscle of P. kl. esculentus. Frog samples were collected at two sites (the Danube-Tisza-Danube canal (DTDC) and the river Ponjavica) in Serbia, which are characterized by different levels of metal pollution. Differences between the metal contents in different tissues showed that the skin of frogs from the DTDC accumulated statistically higher concentrations of Cd, Cu, Pb and Zn, while only the Fe concentration was lower. No significant differences between metal concentrations in muscle tissues of frogs from the DTDC and Ponjavica were observed. Examination of the parameters of the AOS revealed that frogs from the DTDC had higher concentrations of GSH in the liver and of SH groups in the skin and muscle, whereas the activities of the antioxidative enzymes SOD, GHS-Px and GR in the liver and of GR in the skin were lower than in frogs from the Ponjavica. The relationship between metal concentrations and AOS parameters showed the highest number of correlations with GSH, GR and CAT, and with Ni, Zn, Hg, Cr and Cd. Based on the results in this study, we concluded that increased concentrations of heavy metals in frog tissues can alter the AOS, which leads to higher concentrations of GSH and SH groups and lower activities of antioxidative enzymes. The response of the AOS to metal pollutants allowed us to make a distinction between different frog tissues, and to conclude that the liver and skin are more suitable for assessing metal-induced oxidative stress in frogs than muscle.

Sublethal effects of the pyrethroid insecticide deltamethrin on oxidative stress parameters in green toad (Bufotes viridis L.)

Pyrethroids are synthetic insecticides widely used in agriculture, public health, and veterinary medicine. Deltamethrin, a type II pyrethroid insecticide, has attracted particular attention because of its frequent use. The mechanisms of the toxicity of most pesticides (including pyrethroids) in nontarget organisms is linked to the production of free radicals, oxidative stress induction, increased lipid peroxidation, and disruption of the total antioxidant potential. The aim of the present study was to investigate the effects of acute toxicity of 3 different concentrations (8, 16, and 32 mg/kg body wt) of orally applied deltamethrin after 96 h of treatment. Some of the front-line oxidative stress parameters as well as cholinesterase (ChE) activity and cytochrome P4501A (CYP1A) expression in the liver, muscle, skin, and gastrointestinal tissue of the adult green toad Bufotes viridis were determined. Compared with the control group, the activity of catalase and glutathione reductase was increased in the liver and skin, while the concentration of sulfhydryl groups was reduced. In the liver and muscle, concentrations of thiobarbituric reactive substances were increased, as well as liver CYP1A expression. In the muscle and skin, glutathione-S-transferase activity was higher in treated toads. The oxidative stress parameters examined were affected by different deltamethrin concentrations. We conclude that the assessed parameters represent good biomarkers of pesticide-induced oxidative stress. Environ Toxicol Chem 2017;36:2814-2822.

Bioaccumulation and effects of metals on oxidative stress and neurotoxicity parameters in the frogs from the Pelophylax esculentus complex

Metals are involved in the formation of reactive oxygen species and can induce oxidative stress. The aim of this study was to assess the effects of several metals on oxidative stress in the skin and muscle of the Pelophylax esculentus “complex” frogs (parental species Pelophylax ridibundus, Pelophylax lessonae, and their hybrid Pelophylax esculentus) that inhabit the wetland Obedska Bara in Serbia, and the potential use of these species as bioindicator organisms in biomonitoring studies. The biomarkers of oxidative stress (SOD, CAT, GSH-Px, GR, GST activities and GSH, SH concentrations) and cholinesterase activity were investigated. The concentrations of nine metals (Fe, Cu, Zn, As, Cd, Cr, Hg, Ni, and Pb) were measured in the water and tissues. Correlations were established between metals and biomarkers in the tissues. The results of metal accumulation distinguished the skin of P. lessonae and muscle of P. ridibundus from other P. esculentus complex species. The oxidative stress biomarkers observed in P. ridibundus and P. esculentus had greater similarity than in P. lessonae. The P. lessonae displayed the highest number of correlations between biomarkers and metals. The results of tissue responses revealed that skin was more susceptible to metal-induced oxidative stress, with only exception of As. In the light of these findings, we can suggest the use of P. esculentus complex species as a biomonitoring species in studies of metal accumulation and metal-induced oxidative stress, but with special emphasis on P. lessonae.

Extensive mitochondrial heteroplasmy in hybrid water frog (Pelophylax spp.) populations from Southeast Europe.

Abstract Water frogs of the genus Pelophylax (previous Rana) species have been much studied in Europe for their outstanding reproductive mechanism in which sympatric hybridization between genetically distinct parental species produces diverse genetic forms of viable hybrid animals. The most common hybrid is P. esculentus that carries the genomes of both parental species, P. ridibundus and P. lessonae, but usually transfers the whole genome of only one parent to its offsprings (hybridogenesis). The evolutionary cost of transfer of the intact genome and hence the hemiclonal reproduction is the depletion of heterozygosity in the hybrid populations. Pelophylax esculentus presents an excellent example of the long‐term sustained hybridization and hemiclonal reproduction in which the effects of the low genetic diversity are balanced through the novel mutations and periodic recombinations. In this study, we analyzed the mitochondrial (mt) and microsatellites DNA variations in hybrid Pelophylax populations from southern parts of the Pannonian Basin and a north–south transect of the Balkan Peninsula, which are home for a variety of Pelophylax genetic lineages. The mtDNA haplotypes found in this study corresponded to P. ridibundus and P. epeiroticus of the Balkan – Anatolian lineage (ridibundus–bedriagae) and to P. lessonae and a divergent lessonae haplotype of the lessonae lineage. The mtDNA genomes showed considerable intraspecific variation and geographic differentiation. The Balkan wide distributed P. ridibundus was found in all studied populations and its nuclear genome, along with either the lessonae or the endemic epeiroticus genome, in all hybrids. An unexpected finding was that the hybrid populations were invariably heteroplasmic, that is, they contained the mtDNA of both parental species. We discussed the possibility that such extensive heteroplasmy is a result of hybridization and it comes from regular leakage of the paternal mtDNA from a sperm of one species that fertilizes eggs of another. In this case, the mechanisms that protect the egg from heterospecific fertilization and further from the presence of sperm mtDNA could become compromised due to their differences and divergence at both, mitochondrial and nuclear DNA. The heteroplasmy once retained in the fertilized egg could be transmitted by hybrid backcrossing to the progeny and maintained in a population over generations. The role of interspecies and heteroplasmic hybrid animals due to their genomic diversity and better fitness compare to the parental species might be of the special importance in adaptations to miscellaneous and isolated environments at the Balkan Peninsula.

Evidence of Batrachochytrium dendrobatidis Infection in Amphibians from Serbian Lowlands

Abstract We investigated presence of Batrachochytrium dendrobatidis in the Republic of Serbia. Seven out of 88 samples (8%) tested positive, all belonging to the frog genus Pelophylax. Two positive sites were located directly on the Danube River. The Danube River could be an important disease corridor, and distribution of Batrachochytrium dendrobatidis along this river should be further explored.